Project Summary The dramatic rise of antimicrobial resistance has created the need for new approaches in the design of novel drug systems. Amphotericin B (AmB) is an antifungal drug that has been used clinically for over 50 years with only limited cases of clinical resistance reported. Due to the toxic nature of AmB's side effects, alternative antifungals have been developed, but treatment with these alternatives is already generating increasing numbers of resistant strains despite a much shorter period of use. This has led to a renewed interest in the study of AmB as a better understanding of its activity could inform on how to design more effective antimicrobials with lower occurrences of resistance. A wide range of investigations has resulted in three, very different, proposed models for AmB's mechanism of action. We are proposing a series of experiments based on transient absorption microscopy which will image label-free AmB as it interacts with both living and model systems. Our hypotheses are (1) that our proposed methodology will be able to distinguish between the different proposed models of AmB activity and (2) that the mechanism of action by which AmB interacts with a membrane exists as an environmentally driven interplay between the proposed models of ergosterol sequestration, ion channel formation, and oxidative damage. In the first specific aim of this project we will monitor the distribution and orientation of AmB in giant unilamellar vesicles and S. cerevisiae and C. albicans cells. The second specific aim will elucidate the role of aggregation in the mechanism of AmB's interaction. This aggregation is dependent on the environmental conditions making it a likely driver for which model of action AmB employs while also having important implications for the delivery of the drug. Overall, these innovative studies fit well within NIGMS's mission to lay the foundation for advancements in disease treatment and will provide hands-on training and education with cutting-edge imaging instrumentation for four students, including those at both the undergraduate and graduate levels.